Aims: To isolate and evaluate spore-former bacteria for being used as probiotic additives in animal nutrition by their technological features. Study Design: The study was experimental, by using calves’ faeces for spore-forming identification and further evaluation of their “in vitro” probiotic-related properties. Place and Duration of Study: Laboratory of Preventive Microbiology, Centro de Referencia para Lactobacilos (CERELA-CONICET), between June 2013 and November 2013. Methodology: In this work, some Bacillus strains were isolated from calves’ faeces and evaluated for their “in vitro” beneficial characteristics: Surface properties, biosurfactant and emulsification production, and inhibition of pathogens. The antibiotic sensibility was also assayed. Results: Two Bacillus strains were selected, identified by phenotypic and molecular techniques as Bacillus subtilis strains M14 and M12. Spores resulted to be more hydrophobic than vegetative cells. The M14 strain showed biosurfactant and emulsifying properties. Inhibition assays against pathogenic bacteria indicated they inhibit gram-positive microorganisms. The antibiotic susceptibility showed that the two strains were sensitive to the antibiotics assayed, except Bacillus M12 that was resistant to Kanamycin. Conclusion: The results indicate these strains can be further studied for their inclusion in the design of a probiotic product for newborn calves.

The thermosensor histidine kinase DesK from Bacillus subtilis senses changes in membrane fluidity initiating an adaptive response. Structural changes in DesK have been implicated in transmembrane signaling, but direct evidence is still lacking. On the basis of structure-guided mutagenesis, we now propose a mechanism of DesK-mediated signal sensing and transduction. The data indicate that stabilization/destabilization of a 2-helix coiled coil, which connects the transmembrane sensory domain of DesK to its cytosolic catalytic region, is crucial to control its signaling state. Computational modeling and simulations reveal couplings between protein, water and membrane mechanics. We propose that membrane thickening is the main driving force for signal sensing and that it acts by inducing helix stretching and rotation prompting an asymmetric kinase-competent state. Overall, the known structural changes of the sensor kinase, as well as further dynamic rearrangements that we now predict, consistently link structure determinants to activity modulation.

Multicellular biofilm formation and surface motility are bacterial behaviors considered mutually exclusive. However, the basic decision to move over or stay attached to a surface is poorly understood. Here, we discover that in Bacillus subtilis, the key root biofilm-controlling transcription factor Spo0A~Pi (phosphorylated Spo0A) governs the flagellum-independent mechanism of social sliding motility. A Spo0A-deficient strain was totally unable to slide and colonize plant roots, evidencing the important role that sliding might play in natural settings. Microarray experiments plus subsequent genetic characterization showed that the machineries of sliding and biofilm formation share the same main components (i.e., surfactin, the hydrophobin BslA, exopolysaccharide, and de novo-formed fatty acids). Sliding proficiency was transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC. We discovered that potassium, a previously known inhibitor of KinC-dependent biofilm formation, is the specific sliding-activating signal through a thus-far-unnoticed cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. The differential expression of the Spo0A~Pi reporter abrB gene and the different levels of the constitutively active form of Spo0A, Sad67, in Δspo0A cells grown in optimized media that simultaneously stimulate motile and sessile behaviors uncover the spatiotemporal response of KinB and KinC to potassium and the gradual increase in Spo0A~Pi that orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population. Overall, these results provide insights into how multicellular behaviors formerly believed to be antagonistic are coordinately activated in benefit of the bacterium and its interaction with the host.

The thermosensor DesK is a multipass transmembrane histidine-kinase that allows the bacterium Bacillus subtilis to adjust the levels of unsaturated fatty acids required to optimize membrane lipid fluidity. The cytoplasmic catalytic domain of DesK behaves like a kinase at low temperature and like a phosphatase at high temperature. Temperature sensing involves a built-in instability caused by a group of hydrophilic residues located near the N terminus of the first transmembrane (TM) segment. These residues are buried in the lipid phase at low temperature and partially “buoy” to the aqueous phase at higher temperature with the thinning of the membrane, promoting the required conformational change. Nevertheless, the core question remains poorly understood: How is the information sensed by the transmembrane region converted into a rearrangement in the cytoplasmic catalytic domain to control DesK activity? Here, we identify a “linker region” (KSRKERERLEEK) that connects the TM sensor domain with the cytoplasmic catalytic domain involved in signal transmission. The linker adopts two conformational states in response to temperature-dependent membrane thickness changes: (i) random coiled and bound to the phospholipid head groups at the water-membrane interface, promoting the phosphatase state or (ii) unbound and forming a continuous helix spanning a region from the membrane to the cytoplasm, promoting the kinase state. Our results uphold the view that the linker is endowed with a helix/random coil conformational duality that enables it to behave like a transmission switch, with helix disruption decreasing the kinase/phosphatase activity ratio, as required to modulate the DesK output response.

Pink Root is among the major diseases of onion and its causal agent, Setophoma terrestris, is one of the most severe pathogens in soils of tropical and subtropical climates. The management of Pink Root is difficult and the strategies currently used are ineffective. Although, there are some varieties resistant to S. terrestris, they are not resistant to every isolate of the pathogen and it is not unusual to find isolates that break that resistance. Moreover, chemical control is not technically or economically feasible. So, we decided to address biocontrol as a mean to manage the main onion disease in Argentina.We tested the efficiency of a strain of Bacillus subtilis subsp. subtilis isolated from the rhizosphere of onion plants to inhibit the growth of S. terrestris in vitro. Our strain of Bacillus subtilis showed a strong capacity of growth inhibition of S. terrestris. No antagonistic activity against two other onion pathogens, Fusarium oxysporum f. sp. cepae and F. proliferatum, was observed. Interestingly, we found a high growth inhibition of S. terrestris on plates containing cell-free supernatant of B. subtilis previously grown in the presence of the fungus. No significant differences in the fungal growth were obtained between control plates and plates containing cell-free supernatant from B. subtilis grown without previous contact with S. terrestris. Electron microscopy of S. terrestris from co-cultures plates revealed thickened, tortuous or coiledfungal hyphae, with granules and globular like terminations. These results suggested that the strain of B. subtilis under study has a strong biocontrol activity against S. terrestris and that it would be acting diffusible bacterial inhibitory compounds specifically induced by this pathogen.

Pink Root is among the major diseases of onion and its causal agent, Setophoma terrestris, is one of the most severe pathogens in soils of tropical and subtropical climates. The management of Pink Root is difficult and the strategies currently used are ineffective. Although, there are some varieties resistant to S. terrestris, they are not resistant to every isolate of the pathogen and it is not unusual to find isolates that break that resistance. Moreover, chemical control is not technically or economically feasible. So, we decided to address biocontrol as a mean to manage the main onion disease in Argentina. We tested the efficiency of a strain of Bacillus subtilis subsp. subtilis isolated from the rhizosphere of onion plants to inhibit the growth of S. terrestris in vitro. Our strain of Bacillus subtilis showed a strong capacity of growth inhibition of S. terrestris. No antagonistic activity against two other onion pathogens, Fusarium oxysporum f. sp. cepae and F. proliferatum, was observed. Interestingly, we found a high growth inhibition of S. terrestris on plates containing cell-free supernatant of B. subtilis previously grown in the presence of the fungus. No significant differences in the fungal growth were obtained between control plates and plates containing cell-free supernatant from B. subtilis grown without previous contact with S. terrestris. Electron microscopy of S. terrestris from co-cultures plates revealed thickened, tortuous or coiled fungal hyphae, with granules and globular like terminations. These results suggested that the strain of B. subtilis under study has a strong biocontrol activity against S. terrestris and that it would be acting diffusible bacterial inhibitory compounds specifically induced by this pathogen.

Bacillus subtilis spores generally considered safe, have received growing attention due to their potential biotechnological applications including vaccine formulations, particularly as vaccine adjuvants. In the present review we present the status of the adjuvanticity of the spore B. subtilis for mucosal route and our experience regarding its adjuvant activity induced against two model antigens, Tetanus Toxoid (TT) and ovalbumin (Ova) for oral (o.r) and intranasal (i.n) immunization. A document review on B. subtilis, adjuvant, vaccine and mucosal route was carried out in MEDLINE by PubMed, SciELO and LILACS databases. B. subtilis spores (RG 4365) were used for the exploration of the adjuvant activity. Balb/c mice were immunized by i.n and o.r route with TT or Ova combined with B. subtilis spores and specific antibody response in serum, saliva and fecal were measured by ELISA. This review showed the existence of several papers using B. subtilis spores as adjuvant by different methodologies and administration routes, being the expression of recombinant antigens and the the o.r route the most widely used. In our work we found an increase of seric response of IgG, subclass IgG1 and IgG2a and specific IgA in saliva and feces in groups immunized with spores coadministered with Ova and TT by both routes, which was significantly superior to control groups (p<0.05). These data suggest that spores are an efficient mucosal and systemic adjuvant for enhancing humoral immune responses and highlight their clinical potential for future mucosal vaccines.

Autochthonous microorganisms as probiotics are a novel alternative to antibiotics. The genus Bacillus is highly used by its ability to survive on adverse conditions. Piaractus mesopotamicus, a native fish, is the most producedspecie in Argentinian aquaculture. The low level of knowledge, high stress conditions and demand of animals led to a deficit in the number of larvae and fry. The aim of this study was to evaluate the probiotic effect of previously selected autochthonous Bacillus on biometrical parameters of P. mesopotamicus larvae when administered together in a combined suspension in different doses and stages. Results showed that the bacterial administration did not causesignificant effects on the variables evaluated. However, the dose containing 6 × 107 CFU L-1 of each strain showed the highest average values of P. mesopotamicus biological parameters, regardless the stage of administration.Histological evaluations of animals administered with this dose did not showed adverse effects and indicated the capability of this dose, administered in a particularly period of time, to stimulate the early appearance of gastricglands, vestiges of what would be the pylorus and a higher development of liver. Thus, we proposed the safe use ofthis dose in a combined probiotic mixture to be studied in further assays.